Na k atpase α1

Myricitrin was obtained from Aladdin Industrial Corporation (Shanghai, China). Monoclonal and polyclonal antibodies against iNOS, COX-2, JNK, phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JAK1, phospho-JAK1 (Tyr1022/1023), JAK2, phospho-JAK2 (Tyr1007/1008), STAT1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), TBP, gp91^phox, Na/K ATPase-α1, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to p47^phox was obtained from Santa Cruz Biotechnology (CA, USA). All secondary antibodies used for western blotting were purchased from LI-COR Biosciences (Lincoln, NE, USA). LPS (from Escherichia coli 0111:B4), NAC, and DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 was purchased from KeyGen Biotech (Nanjing, JS, China). CM-H2DCFDA was obtained from Invitrogen (Carlsbad, CA, USA). Ruxolitinib and apocynin were purchased from Selleck Chemicals (Houston, TX, USA). All ELISA kits were purchased from R&D Systems China Co. Ltd. (Shanghai, China).

Mouse and rat LV tissue was harvested and lysed. Protein concentrations were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., 23227). Proteins were separated by electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris-buffered saline containing Tween-20 (pH 7.6) and 5% nonfat dry milk for 2 h, and subsequently incubated overnight at 4 °C with primary antibodies to the following proteins: Bcl-2 (#3498), Bax (#5023), GRP78 (#3177), caspase-12 (#2202), PKA (#4782), p-PKA (Thr197), AMPK (#2532), p-AMPK (Thr172, #2531), CREB (#9197), p-CREB (Ser133, #9198), Akt (#2967), p-Akt (Thr308, #13038), ERK1/2 (#4696), p-ERK1/2 (Thr202/Tyr204, #9106), Na⁺-K⁺-ATPase α1 (#23565), β-actin (#8457) (all from Cell Signaling Technology, Inc.), CTRP9 (LifeSpan Biosciences, Inc., LS-C373857), CRT (Abcam, ab22683 and Santa Cruz, sc-373863), AdiopR1 (Abcam, ab126611), Calnexin (Santa Cruz, sc-23954), KDEL ER marker (Santa Cruz, sc-58774), and GAPDH (CMCTAG, Inc., AT0002). After washing, the membranes were probed with appropriate secondary antibodies (Zhongshan Company, ZB-2301, ZB-2305) at room temperature for 90 min. Protein bands were detected by Bio-Rad Imaging System (Hercules), and normalized to β-actin or GAPDH.

Na, K-ATPase α1 (Cell Signaling, 23565), ENPP1 (Cell Signaling, 5342), Caveolin (Cell Signaling, 3267), 4F2hc/CD98 (Cell Signaling, 47213), Pan-Cadherin (Cell Signaling, 4073), Clathrin heavy chain (Cell Signaling, 4796), EEA1 (Cell Signaling, 3288), Rab5a (Cell Signaling, 46449), Rab7 (Cell Signaling, 9367), Rab11 (Cell Signaling, 5589), Calnexin (Cell Signaling, 2679), ERp72 (Cell Signaling, 5033), PDI (Cell Signaling, 3501), RCAS1 (Cell Signaling, 12290), Syntaxin-6 (Cell Signaling, 2869), Anti-rabbit IgG-HRP (Cell Signaling, 7074), Anti-mouse IgG-HRP (Cell Signaling, 7076).
Dignam lysates were prepared as per the procedure mentioned earlier (Dignam et al.,1983) with minor modifications. Cell pellets were collected, washed twice in ice-cold phosphate-buffered saline (PBS) and resuspended at one million cells per 0.5 mL of buffer C (0.42 M NaCl, 10% glycerol, 20 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) (14 (link)), followed by centrifugation for 15 min at 10,000 g.